-
Frontiers in Microbiomes 2022Organophosphate nerve agents (OPNAs) act as irreversible inhibitors of acetylcholinesterase and can lead to cholinergic crisis including salivation, lacrimation,...
Organophosphate nerve agents (OPNAs) act as irreversible inhibitors of acetylcholinesterase and can lead to cholinergic crisis including salivation, lacrimation, urination, defecation, gastrointestinal distress, respiratory distress, and seizures. Although the OPNAs have been studied in the past few decades, little is known about the impact on the gut microbiome which has become of increasing interest across fields. In this study, we challenged animals with the OPNA, diisopropylfluorophosphate (DFP, 4mg/kg, s.c.) followed immediately by 2mg/kg atropine sulfate (i.m.) and 25mg/kg 2-pralidoxime (i.m.) and 30 minutes later by 3mg/kg midazolam (i.m.). One hour after midazolam, animals were treated with a dosing regimen of saracatinib (SAR, 20mg/kg, oral), a src family kinase inhibitor, to mitigate DFP-induced neurotoxicity. We collected fecal samples 48 hours, 7 days, and 5 weeks post DFP intoxication. 16S rRNA genes (V4) were amplified to identify the bacterial composition. At 48 hours, a significant increase in the abundance of and decrease in the abundance of were observed in DFP treated animals. At 7 days there was a significant reduction in and , but a significant increase in in the DFP groups compared to controls. The taxonomic changes at 5 weeks were negligible. There was no impact of SAR administration on microbial composition. There was a significant DFP-induced reduction in alpha diversity at 48 hours but not at 7 days and 5 weeks. There appeared to be an impact of DFP on beta diversity at 48 hours and 7 days but not at 5 weeks. In conclusion, acute doses of DFP lead to short-term gut dysbiosis and SAR had no effect. Understanding the role of gut dysbiosis in long-term toxicity may reveal therapeutic targets.
PubMed: 37304619
DOI: 10.3389/frmbi.2022.1006078 -
Biomedicine & Pharmacotherapy =... Feb 2022Age-related meibomian gland dysfunction (MGD) is the main cause of evaporative dry eye disease in an aging population. Decreased meibocyte cell renewal and lipid...
Age-related meibomian gland dysfunction (MGD) is the main cause of evaporative dry eye disease in an aging population. Decreased meibocyte cell renewal and lipid synthesis are associated with age-related MGD. Here, we found an obvious decline of Ki67, ΔNp63, and Na/K ATPase expression in aged meibomian glands. Potential Na/K ATPase agonist periplocin, a naturally occurring compound extracted from the traditional herbal medicine cortex periplocae, could promote the proliferation and stem cell activity of meibocyte cells in vitro. Moreover, we observed that periplocin treatment effectively increased the expression of Na+ /K+ ATPase, accompanied with the enhanced expression of Ki67 and ΔNp63 in aged meibomian glands, indicating that periplocin may accelerate meibocyte cell renewal in aged mice. LipidTox staining showed increased lipid accumulation after periplocin treatment in cultured meibomian gland cells and aged meibomian glands. Furthermore, we demonstrated that the SRC pathway was inhibited in aged meibomian glands; however, it was activated by periplocin. Accordingly, the inhibition of the SRC signaling pathway by saracatinib blocked periplocin-induced proliferation and lipid accumulation in meibomian gland cells. In sum, we suggest periplocin-ameliorated meibocyte cell renewal and lipid synthesis in aged meibomian glands via the SRC pathway, which could be a promising candidate for age-related MGD.
Topics: Aging; Animals; Cell Proliferation; Cells, Cultured; Epithelial Cells; Ki-67 Antigen; Male; Meibomian Gland Dysfunction; Meibomian Glands; Mice, Inbred C57BL; Saponins; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Up-Regulation; src-Family Kinases; Mice
PubMed: 34883449
DOI: 10.1016/j.biopha.2021.112487 -
The Journal of Headache and Pain Dec 2021Purinergic P2X7 receptor plays an important role in migraine pathophysiology. Yet precise molecular mechanism underlying P2X7R signaling in migraine remains unclear....
BACKGROUND
Purinergic P2X7 receptor plays an important role in migraine pathophysiology. Yet precise molecular mechanism underlying P2X7R signaling in migraine remains unclear. This study explores the hypothesis that P2X7 receptor transmits signaling to Src family kinases (SFKs) during cortical spreading depression (CSD) and neuroinflammation after CSD.
METHODS
CSD was recorded using electrophysiology in rats and intrinsic optical imaging in mouse brain slices. Cortical IL-1β and TNFα mRNA levels were detected using qPCR. Glutamate release from mouse brain slices was detected using glutamate assay.
RESULTS
The data showed that deactivation of SFKs by systemic injection of PP2 reduced cortical susceptibility to CSD in rats and CSD-induced IL-1β and TNF-α gene expression in rat ipsilateral cortices. Consistently, in mouse brain slices, inhibition of SFKs activity by saracatinib and P2X7 receptor by A740003 similarly reduced cortical susceptibility to CSD. When the interaction of P2X7 receptor and SFKs was disrupted by TAT-P2X7, a marked reduction of cortical susceptibility to CSD, IL-1β gene expression and glutamate release after CSD induction were observed in mouse brain slices. The reduced cortical susceptibility to CSD by TAT-P2X7 was restored by NMDA, and disrupting the Fyn-NMDA interaction using TAT-Fyn (39-57) but not disrupting Src-NMDA receptor interaction using TAT-Src (40-49) reduced cortical susceptibility to CSD. Furthermore, activation of P2X7 receptor by BzATP restored the TAT-Fyn (39-57)-reduced cortical susceptibility to CSD.
CONCLUSION
This study reveals that SFKs activity transmits P2X7 receptor signaling to facilitate CSD propagation via glutamatergic pathway and promote neuroinflammation, which is of particular relevance to migraine.
Topics: Animals; Cortical Spreading Depression; Mice; Neuroinflammatory Diseases; Rats; Receptors, Purinergic P2X7; Signal Transduction; src-Family Kinases
PubMed: 34863113
DOI: 10.1186/s10194-021-01359-8 -
Nature Communications Dec 2021mRNA expression of the DLC1 tumor suppressor gene is downregulated in many lung cancers and their derived cell lines, with DLC1 protein levels being low or absent....
mRNA expression of the DLC1 tumor suppressor gene is downregulated in many lung cancers and their derived cell lines, with DLC1 protein levels being low or absent. Although the role of increased EZH2 methyltransferase in cancer is usually attributed to its histone methylation, we unexpectedly observed that post-translational destabilization of DLC1 protein is common and attributable to its methylation by cytoplasmic EZH2, leading to CUL-4A ubiquitin-dependent proteasomal degradation of DLC1. Furthermore, siRNA knockdown of KRAS in several lines increases DLC1 protein, associated with a drastic reduction in cytoplasmic EZH2. Pharmacologic inhibition of EZH2, CUL-4A, or the proteasome can increase the steady-state level of DLC1 protein, whose tumor suppressor activity is further increased by AKT and/or SRC kinase inhibitors, which reverse the direct phosphorylation of DLC1 by these kinases. These rational drug combinations induce potent tumor growth inhibition, with markers of apoptosis and senescence, that is highly dependent on DLC1 protein.
Topics: Animals; Antineoplastic Agents; Benzodioxoles; Boron Compounds; Cell Line, Tumor; Enhancer of Zeste Homolog 2 Protein; GTPase-Activating Proteins; Gene Knockdown Techniques; Gene Knockout Techniques; Glycine; HEK293 Cells; Heterocyclic Compounds, 3-Ring; Humans; Lung Neoplasms; Mice; Mutagenesis, Site-Directed; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinase Inhibitors; Protein Stability; Proto-Oncogene Proteins p21(ras); Quinazolines; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays
PubMed: 34862367
DOI: 10.1038/s41467-021-26993-3 -
International Journal of Molecular... Nov 2021Transient receptor potential ankyrin 1 (TRPA1) plays a role in migraine and is proposed as a promising target for migraine therapy. However, TRPA1-induced signaling in...
Transient receptor potential ankyrin 1 (TRPA1) plays a role in migraine and is proposed as a promising target for migraine therapy. However, TRPA1-induced signaling in migraine pathogenesis is poorly understood. In this study, we explored the hypothesis that Src family kinases (SFKs) transmit TRPA1 signaling in regulating cortical spreading depression (CSD), calcitonin gene-related peptide (CGRP) release and neuroinflammation. CSD was monitored in mouse brain slices via intrinsic optical imaging, and in rats using electrophysiology. CGRP level and IL-1β gene expression in mouse trigeminal ganglia (TG) was detected using Enzyme-linked Immunosorbent Assay and Quantitative Polymerase Chain Reaction respectively. The results showed a SFKs activator, pYEEI (EPQY(PO3H2)EEEIPIYL), reversed the reduced cortical susceptibility to CSD by an anti-TRPA1 antibody in mouse brain slices. Additionally, the increased cytosolic phosphorylated SFKs at Y416 induced by CSD in rat ipsilateral cerebral cortices was attenuated by pretreatment of the anti-TRPA1 antibody perfused into contralateral ventricles. In mouse TG, a SFKs inhibitor, saracatinib, restored the CGRP release and IL-1β mRNA level increased by a TRPA1 activator, umbellulone. Moreover, umbellulone promoted SFKs phosphorylation, which was reduced by a PKA inhibitor, PKI (14-22) Amide. These data reveal a novel mechanism of migraine pathogenesis by which TRPA1 transmits signaling to SFKs via PKA facilitating CSD susceptibility and trigeminovascular system sensitization.
Topics: Animals; Calcitonin Gene-Related Peptide; Cerebral Cortex; Cortical Spreading Depression; Electrophysiology; Gene Expression; Interleukin-1beta; Male; Mice, Inbred C57BL; Migraine Disorders; Neuroglia; Neurons; RNA, Messenger; Rats, Sprague-Dawley; TRPA1 Cation Channel; Trigeminal Ganglion; src-Family Kinases; Mice; Rats
PubMed: 34830154
DOI: 10.3390/ijms222212273 -
Bone Jan 2022To observe the effect of AZD0530 on the progression of knee OA after blocking β-catenin phosphorylation and then dormancy of the Wnt/β pathway by tyrosine kinase Fyn.
OBJECTIVE
To observe the effect of AZD0530 on the progression of knee OA after blocking β-catenin phosphorylation and then dormancy of the Wnt/β pathway by tyrosine kinase Fyn.
METHODS
The levels of Fyn, β-catenin, p-β-catenin (Tyr142), the chondrocyte positive marker Aggrecan, and the chondrocyte negative marker MMP13 were observed in human knee tibial plateau chondrocytes in vivo and in vitro. Different doses of AZD0530 were used to treat chondrocytes of the human OA tibial plateau chondrocytes in vitro, and the degree of chondrocyte degeneration was observed. Different doses of AZD0530 were intraarticularly injected into OA rats to observe the degree of tibial plateau cartilage degeneration.
RESULTS
When OA occurred in human knee, the levels of tyrosine kinase Fyn,β-catenin and p-β-catenin (Tyr142) in chondrocytes increased significantly.The level of Aggrecan decreased and MMP13 increased in chondrocytes. The levels of β-catenin, p-β-catenin (Tyr142) and MMP13 in chondrocytes decreased, while the level of Aggrecan increased after AZD0530 was used to intervene chondrocytes in vitro, which was positively correlated with the dose of AZD0530. Intra-articular injection of AZD0530 obviously attenuated the degeneration of articular cartilage, which was positively correlated with the dose of AZD0530.
CONCLUSION
The level of Fyn in chondrocytes of human knee tibial plateau increased significantly when OA occurred. AZD0530 can inhibit tyrosine kinase Fyn from β-catenin phosphorylation, a key Wnt/β pathway protein, and then inhibit Wnt/β pathway levels in chondrocytes. This finding also suggests that disruption of the Wnt/β pathway with AZD0530 provides chondral protection in rat posttraumatic OA.
Topics: Animals; Benzodioxoles; Cartilage, Articular; Cells, Cultured; Chondrocytes; Osteoarthritis; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-fyn; Quinazolines; Rats; Wnt Signaling Pathway; beta Catenin
PubMed: 34798298
DOI: 10.1016/j.bone.2021.116259 -
Frontiers in Cellular Neuroscience 2021Acute organophosphate (OP) toxicity poses a significant threat to both military and civilian personnel as it can lead to a variety of cholinergic symptoms including the...
Differential Impact of Severity and Duration of , Medical Countermeasures, and a Disease-Modifier, Saracatinib, on Brain Regions in the Rat Diisopropylfluorophosphate Model.
Acute organophosphate (OP) toxicity poses a significant threat to both military and civilian personnel as it can lead to a variety of cholinergic symptoms including the development of (SE). Depending on its severity, SE can lead to a spectrum of neurological changes including neuroinflammation and neurodegeneration. In this study, we determined the impact of SE severity and duration on disease promoting parameters such as gliosis and neurodegeneration and the efficacy of a disease modifier, saracatinib (AZD0530), a Src/Fyn tyrosine kinase inhibitor. Animals were exposed to 4 mg/kg diisopropylfluorophosphate (DFP, s.c.) followed by medical countermeasures. We had five experimental groups: controls (no DFP), animals with no continuous convulsive seizures (CS), animals with ∼20-min continuous CS, 31-60-min continuous CS, and > 60-min continuous CS. These groups were then assessed for astrogliosis, microgliosis, and neurodegeneration 8 days after DFP exposure. The 31-60-min and > 60-min groups, but not ∼20-min group, had significantly upregulated gliosis and neurodegeneration in the hippocampus compared to controls. In the piriform cortex and amygdala, however, all three continuous CS groups had significant upregulation in both gliosis and neurodegeneration. In a separate cohort of animals that had ∼20 and > 60-min of continuous CS, we administered saracatinib for 7 days beginning three hours after DFP. There was bodyweight loss and mortality irrespective of the initial SE severity and duration. However, in survived animals, saracatinib prevented spontaneous recurrent seizures (SRS) during the first week in both severity groups. In the ∼20-min CS group, compared to the vehicle, saracatinib significantly reduced neurodegeneration in the piriform cortex and amygdala. There were no significant differences in the measured parameters between the naïve control and saracatinib on its own (without DFP) groups. Overall, this study demonstrates the differential effects of the initial SE severity and duration on the localization of gliosis and neurodegeneration. We have also demonstrated the disease-modifying potential of saracatinib. However, its' dosing regimen should be optimized based on initial severity and duration of CS during SE to maximize therapeutic effects and minimize toxicity in the DFP model as well as in other OP models such as soman.
PubMed: 34720886
DOI: 10.3389/fncel.2021.772868 -
Pharmaceuticals (Basel, Switzerland) Sep 2021Osteosarcoma (OS) is an aggressive mesenchymal cell tumor that carries a poor long-term prognosis. Despite definitive surgery for the primary tumor and adjuvant...
Osteosarcoma (OS) is an aggressive mesenchymal cell tumor that carries a poor long-term prognosis. Despite definitive surgery for the primary tumor and adjuvant chemotherapy, pulmonary metastasis is common and is the primary cause of morbidity. To improve outcomes for patients, we have developed and optimized a phenotypic screen for drugs that may target OS disseminated tumor cells (DTCs) and inhibit their metastatic outbreak rather than merely screening for cytotoxic activity against proliferating cells, as is commonly conducted in conventional drug discovery approaches. We report on the validation of a previously described 3D reconstituted basement membrane extract (3D BME) model system for tumor dormancy and metastatic outgrowth adapted to clonal pairs of high and low metastatic OS cells. A post-hoc validation of the assay was possible by comparing the activity of a drug in our assay with early evidence of activity in human OS clinical trials (regorafenib and saracatinib). In this validation, we found concordance between our assay and human clinical trial experience We then explored an approved veterinary small molecule inhibitor of Janus kinase-1 (oclacitinib) as a potential drug candidate to take advantage of the high prevalence of OS in pet dogs and its translational value to humans. Despite the biological rationale, we found no evidence to support the use of oclacitinib as an antimetastatic agent in OS. The findings support our 3D BME assay as a highly efficient method to examine drugs for activity in targeting OS DTCs.
PubMed: 34681195
DOI: 10.3390/ph14100971 -
Bioengineering & Translational Medicine Sep 2021Macrophages are generally thought to play a key role in the pathogenesis of aseptic loosening through initiating periprosthetic inflammation and pathological bone...
Macrophages are generally thought to play a key role in the pathogenesis of aseptic loosening through initiating periprosthetic inflammation and pathological bone resorption. The aim of this study was to identify macrophage-derived factors that promote osteoclast differentiation and periprosthetic bone destruction. To achieve this, we examined the effects of 12 macrophage-derived factors that were identified by RNA-seq analysis of stimulated macrophages on osteoclast differentiation. Surprisingly, thymidine phosphorylase (TYMP) was found to trigger significant number of osteoclasts that exhibited resorbing activities on dentine slices. Functionally, TYMP knockdown reduced the number of osteoclasts in macrophages that had been stimulated with polyethylene debris. TYMP were detected in serum and synovial tissues of patients that had been diagnosed with aseptic loosening. Moreover, the administration of TYMP onto calvariae of mice induced pathological bone resorption that was accompanied by an excessive infiltration of inflammatory cells and osteoclasts. The RNA-seq for TYMP-induced-osteoclasts was then performed in an effort to understand action mode of TYMP. TYMP stimulation appeared to activate the tyrosine kinase FYN signaling associated with osteoclast formation. Oral administration of saracatinib, a FYN kinase inhibitor, significantly suppressed formation of bone osteolytic lesions in a polyethylene debris-induced osteolysis model. Our findings highlight a novel molecular target for therapeutic intervention in periprosthetic osteolysis.
PubMed: 34589604
DOI: 10.1002/btm2.10232 -
IScience Sep 2021Resistance to current therapies is common for pancreatic cancer and hence novel treatment options are urgently needed. In this work, we developed and validated a...
Resistance to current therapies is common for pancreatic cancer and hence novel treatment options are urgently needed. In this work, we developed and validated a computational method to select synergistic compound combinations based on transcriptomic profiles from both the disease and compound side, combined with a pathway scoring system, which was then validated prospectively by testing 30 compounds (and their combinations) on PANC-1 cells. Some compounds selected as single agents showed lower GI50 values than the standard of care, gemcitabine. Compounds suggested as combination agents with standard therapy gemcitabine based on the best performing scoring system showed on average 2.82-5.18 times higher synergies compared to compounds that were predicted to be active as single agents. Examples of highly synergistic in vitro validated compound pairs include gemcitabine combined with Entinostat, thioridazine, loperamide, scriptaid and Saracatinib. Hence, the computational approach presented here was able to identify synergistic compound combinations against pancreatic cancer cells.
PubMed: 34585118
DOI: 10.1016/j.isci.2021.103080